Cyclin-Dependent Kinases and CDC7 as Therapeutic Targets Geoffrey Shapiro, M.D., Ph.D. Dana-Farber Cancer Institute Gary K. Schwartz, M.D. Columbia University
Disclosures Geoffrey Shapiro, MD, PhD Advisory Board: G1 Therapeutics, Syros, Vertex, Millenium Research Funding: Pfizer Funding to Dana-Farber Cancer Institute: Pfizer Lilly Novartis Merck Cyclacel Bayer Investigator: palbociclib, abemaciclib, ribociclib, dinaciclib, seliciclib, roniciclib
Disclosures Gary K. Schwartz, MD Consultant for and honoraria received from: Novartis Astra-Zeneca Boehringer
Cell Cycle and Transcriptional CDKs Malumbres. Genome Biology 2014 15:122
Model of CDK Activation Malumbres. Genome Biology 2014 15:122
Overview of CDK Function Malumbres. Genome Biology 2014 15:122
CDK Inhibitor Compounds CDK4/6 Selective Palbociclib (PD0332991) Abemaciclib (LY2835219) Ribociclib (LEE011) CDK 1/2/9 or pan-cdk (+CDK 4/5/7) Flavopiridol Dinaciclib (SCH727965) Seliciclib (CYC202) CYC065 Roniciclib (BAY1000394) CDK7 THZ1 (covalent)
Key Issues for CDK4/6 Inhibitors What governs a G1 arrest response vs. cytotoxic response? Solid tumor models tend to undergo G1 arrest, whereas abrupt apoptosis has been observed in models of T Cell Leukemia and Burkitt Lymphoma. CDK6 is a chromatin-bound NF-κB co-factor. Among cell types that undergo initial G1 arrest, what governs a senescent response? HER2-amplified BC, ER+ BC and KRAS-mutant NSCLC likely undergo senescence following CDK4/6 inhibition, possibly with associated tumor regression that can occur late. CDK4/6 inhibition destabilizes FOXM1, which suppresses senescence. In KRAS-mutant NSCLC, does the tumor suppressive background matter (KRAS alone, KRAS/TP53, KRAS/LKB1, KRAS/CDKN2A/B)?
Key Issues for CDK4/6 Inhibitors What are mechanisms of resistance to CDK4/6 inhibitors? De novo resistance linked to Rb-negativity Acquired resistance linked to CDK2 activation (Cyclin E overexpression or p27 Kip1 loss). What are the most compelling combinations for future development? Hormonal Therapy in ER+ breast cancer (Basis of initial palbociclib US FDA approval in first-line metastatic ER+ breast cancer) MEK inhibition in RAS-driven cancers PI3K inhibition in tumors with PI3K pathway activation Other signal transduction inhibitors (IGF-1R) Chemotherapy (e.g. Taxane) when appropriately sequenced
Palbociclib Induces G1 Cell Cycle Arrest in all Liposarcoma Cell Lines Kovatcheva M et al, Oncotarget, 2015
Palbociclib induces senescence in some but not all RB+ liposarcoma cell lines Senescence Non-Senescence senescence-associated β- galactosidase Senescence Non-Senescence HPγ1 staining for senescence-associated heterochromatic foci formation Kovatcheva M et al, Oncotarget, 2015
shcdk4 Replicates the Effect of Palbociclib Senescence Non-Senescence Kovatcheva M et al, Oncotarget, 2015
Palbociclib or shcdk4 Suppresses MDM2 Expression in Senescent Liposarcoma Cells B Δ by densitometry 0.47 0.24 1.03 0.74 Senescence Non-Senescence Kovatcheva M et al, Oncotarget, 2015
MDM2 Knockdown Induces Cellular Senescence in All Liposaracoma Cells Senescence Non-Senescence Kovatcheva M et al, Oncotarget, 2015
Palbociclib Induces MDM2 Knockdown and Senescence in GBM and Breast Cancer Cells Independent of p53 Status GBM Breast Cancer Kovaatcheva et al, Oncotarget, 2015
MDM2 Loss is Associated with Prolonged Clinical Benefit in Liposarcoma Patients Treated with Palbociclib Kovatcheva M et al, Oncotarget, 2015
ATRX is Required for Palbocilcib-mediated MDM2 loss and Induction of Senescence c C. D Senescence CC Non-Senescence
Model of MDM2 Regulation by ATRX to Induce Senescence CDK4 inhibition causes dissociation of HAUSP/USP7 from MDM2, priming it for autoubiquitination; (2) Degradation of MDM2 also requires ATRX; (3) MDM2 likely ubiquinates a SAP other than p53; removal of MDM2 promotes senescence. Kovatcheva M et al, Oncotarget, 2015
CDK2 as a target Replacement of p27 Kip1 Dominant negative mutants CDK2 inhibitory peptides Targeted cyclin A degradation CDK2 anti-sense and sirna experiments cdk2-/- knockout cells
Combined CDK1/2 depletion is required for antiproliferative effects Cai et al. Cancer Research, 2006
Putative CDK2-dependent tumors: HGSOC
Ovarian and TNBCs BRCA-deficient/ HR-deficient Cisplatin sensitive PARP inhibitor sensitive based on synthetic lethality of PARP inhibition and HR deficiency CCNE1-amplified/ HR-dependent High cyclin E expression Replicative stress upregulates HR genes and HR dependency Often rapid recurrence and platinum-insensitive tumors Likely CDK2 dependent CDK1/CDK2 required for initiation of HR CDK2 and 1 targeting agents also inhibit transcriptional CDKs to varying degrees
Cell Cycle Kinases Malumbres Physiol Rev 2011; 91: 973-1007
CDC7 and CDK in DNA Replication Labib. Genes Dev 2010; 24: 1208-19
CDC7 in Translesion Synthesis Yamada et al. Cell Cycle 2014; 13: 1859-66
RNA Pol II CTD Phosphorylation Heidemann et al. BBA 2013; 1829: 55-62
CDK-dependent regulation of initiation to elongation switch of RNA polymerase II Larochelle et al. Nat Struct Mol Biol 2012; 19: 1108-1115
Concept of Super-enhancers Typical enhancers composed of transcription factor binding sites located at a distance from the transcriptional start site that act through chromosomal looping events to enhance transcription. Super-enhancers consist of very large clusters of enhancers that are densely occupied by transcription factors, co-factors and chromatin regulators (e.g. BRD4) arise via gene amplification, translocation or transcription factor overexpression facilitate high level of expression of genes involved in cell identity, growth and proliferation; often genes and encoded proteins have short half-life, so high-level transcription is critical to maintenance of their expression highly sensitive to perturbation Whyte et al. Cell 2013; 153: 307-19; Lovén et al. Cell 2013; 153: 320-34; Chapuy et al. Cancer Cell 2013; 24: 777-90.
Example of MYC Super-enhancer THZ1 is a covalent CDK7 inhibitor-drug bind with a unique cysteine residue outside of the kinase domain results in prolonged and irreversible CDK7 inactivation BRD4 inhibition (bromodomain Inhibitor) expected to produce similar effects CDK9 inhibitor may also produce similar effects; known CDK9 inhibitors (flavopiridol, Dinaciclib) are ATPcompetitive and reversible Kwiatkowski et al. Nature 2014; 511: 616-20; Chipumuro et al. Cell 2014; 159: 1126-39; Christensen et al. Cancer Cell 2014; 26: 909-22
Concept of Super-enhancers Lovén et al. Cell 2013; 153: 320-34
Transcriptional CDK depletion increases cytotoxicity following CDK1/2 depletion Cai et al. Cancer Research, 2006
CDK12 is a CTD Kinase that regulates transcriptional elongation of a small subset of genes Pathway analysis indicates preferential loss of genes involved in DNA replication, recombination and repair (e.g. BRCA1, ATR, FANCI, FANCD2) Blazec et al. Genes Dev 2011: 25: 2158-72
CDK12 causes defects in homologous recombination repair Bajrami et al. Cancer Res 2014: 74: 287-97
Loss of CDK12 Activity in a Subset of Ovarian Tumors TCGA, Nature 2011
CDKs in the DNA Damage Response CDK Family Member CDK1 CDK2 CDK9 CDK12 CDK5 CDK4/6 BRCA1, CtIP Target ATRIP, CtIP, {BRCA2} RNA Polymerase II (RAD51) RNA Polymerase II (DDR cassette) Sensitization to PARP inhibition in sirna screen G1 arrest and reliance on NHEJ
Cell Cycle and Transcriptional CDK Inhibitors: Summary CDK4/6 inhibitors have anti-tumor activity; additional work needed on biological outcomes in a variety of tumor backgrounds, mechanisms of resistance and development of optimal combinations CDK2 is easily compensated by CDK1; CDK2-dependent tumors may exist (e.g. subset of HGSOCs), requiring clinical validation CDK7, BRD4 and MYC all can overcome transcriptional pausing and activate or recruit CDK9 or 12 for productive transcriptional elongation Targeting CDK7 or BRD4 has greatest effect on super-enhancerdirected transcription and so may be relatively selective for transformed cells CDK12 is involved in the transcription of DDR genes; mutations confer homologous recombination repair deficiency in ovarian cancer cells